In the description, the following terms are employed:
Analyte—A substance or substances, either alone or in admixtures, whose presence is to be detected and, if desired, quantitated. The analyte may be a DNA or RNA molecule of small or high molecular weight, a molecular complex including those molecules, or a biological system containing nucleic acids, such as a virus, a cell, or group of cells. Among the common analytes are nucleic acids (DNA and RNA) or segments thereof, oligonucleotides, either single- or double-stranded, viruses, bacteria, cells in culture, and the like. Bacteria, either whole or fragments thereof, including both gram positive and gram negative bacteria, fungi, algae, and other microorganisms are also analytes, as well as animal (e.g., mammalian) and plant cells and tissues.
Probe—A labelled polynucleotide or oligonucleotide sequence which is complementary to a polynucleotide or oligonucleotide sequence of a particular analyte and which hybridizes to said analyte sequence.
Label—That moiety attached to a polynucleotide or oligonucleotide sequence which comprises a signalling moiety capable of generating a signal for detection of the hybridized probe and analyte. The label may consist only of a signalling moiety, e.g., an enzyme attached directly to the sequence. Alternatively, the label may be a combination of a covalently attached bridging moiety and signalling moiety or a combination of a non-covalently bound bridging moiety and signalling moiety which gives rise to a signal which is detectable, and in some cases quantifiable.
Bridging Moiety—That portion of a label which on covalent attachment or non-covalent binding to a polynucleotide or oligonucleotide sequence acts as a link or a bridge between that sequence and a signalling moiety.
Signalling Moiety—That portion of a label which on covalent attachment or non-covalent binding to a polynucleotide or oligonucleotide sequence or to a bridging moiety attached or bound to that sequence provides a signal for detection of the label.
Signal—That characteristic of a label or signalling moiety that permits it to be detected from sequences that do not carry the label or signalling moiety.
The analysis and detection of minute quantities of substances in biological and non-biological samples has become a routine practice in clinical, diagnostic and analytical laboratories. These detection techniques can be divided into two major classes: (1) those based on ligand-receptor interactions (e.g., immunoassay-based techniques), and (2) those based on nucleic acid hybridization (polynucleotide sequence-based techniques).
Immunoassay-based techniques are characterized by a sequence of steps comprising the non-covalent binding of an antibody and antigen complementary to it. See, for example, T. Chard, An Introduction To Radioimmunoassay And Related Techniques (1978).
Polynucleotide sequence-based detection techniques are characterized by a sequence of steps comprising the non-covalent binding of a labelled polynucleotide sequence or probe to a complementary sequence of the analyte under hybridization conditions in accordance with the Watson-Crick base pairing of adenine (A) and thymine (T), and guanine (G) and cytosine (C), and the detection of that hybridization. [M. Grunstein and D. S. Hogness, “Colony Hybridization: A Method For The Isolation Of Cloned DNAs That Contain A Specific Gene”, Proc. Natl. Acad. Sci. USA, 72, pp. 3961–65 (1975)]. Such polynucleotide detection techniques can involve a fixed analyte [see, e.g., U.S. Pat. No. 4,358,535 to Falkow et al], or can involve detection of an analyte in solution [see U.K. patent application 2,019,408 A].
The primary recognition event of polynucleotide sequence-based detection techniques is the non-covalent binding of a probe to a complementary sequence of an analyte, brought about by a precise molecular alignment and interaction of complementary nucleotides of the probe and analyte. This binding event is energetically favored by the release of non-covalent bonding free energy, e.g., hydrogen bonding, stacking free energy and the like.
In addition to the primary recognition event, it is also necessary to detect when binding takes place between the labelled polynucleotide sequence and the complementary sequence of the analyte. This detection is effected through a signalling step or event. A signalling step or event allows detection in some quantitative or qualitative manner, e.g., a human or instrument detection system, of the occurrence of the primary recognition event.
The primary recognition event and the signalling event of polynucleotide sequence based detection techniques may be coupled either directly or indirectly, proportionately or inversely proportionately. Thus, in such systems as nucleic acid hybridizations with sufficient quantities of radiolabeled probes, the amount of radio-activity is usually directly proportional to the amount of analyte present. Inversely proportional techniques include, for example, competitive immuno-assays, wherein the amount of detected signal decreases with the greater amount of analyte that is present in the sample.
Amplification techniques are also employed for enhancing detection wherein the signalling event is related to the primary recognition event in a ratio greater than 1:1. For example, the signalling component of the assay may be present in a ratio of 10:1 to each recognition component, thereby providing a 10-fold increase in sensitivity.
A wide variety of signalling events may be employed to detect the occurrence of the primary recognition event. The signalling event chosen depends on the particular signal that characterizes the label or signalling moiety of the polynucleotide sequence employed in the primary recognition event. Although the label may only consist of a signalling moiety, which may be detectable, it is more usual for the label to comprise a combination of a bridging moiety covalently or non-covalently bound to the polynucleotide sequence and a signalling moiety that is itself detectable or that becomes detectable after further modification.
The combination of bridging moiety and signalling moiety, described above, may be constructed before attachment or binding to the sequence, or it may be sequentially attached or bound to the sequence. For example, the bridging moiety may be first bound or attached to the sequence and then the signalling moiety combined with that bridging moiety. In addition, several bridging moieties and/or signalling moieties may be employed together in any one combination of bridging moiety and signalling moiety.
Covalent attachment of a signalling moiety or bridging moiety/signalling moiety combination to a sequence is exemplified by the chemical modification of the sequence with labels comprising radioactive moieties, fluorescent moieties or other moieties that themselves provide signals to available detection means or the chemical modification of the sequence with at least one combination of bridging moiety and signalling moiety to provide that signal.
Non-covalent binding of a signalling moiety or bridging moiety/signalling moiety to a sequence involve the non-covalent binding to the sequence of a signalling moiety that itself can be detected by appropriate means, i.e., or enzyme, or the non-covalent binding to the sequence of a bridging moiety/signalling moiety to provide a signal that may be detected by one of those means. For example, the label of the polynucleotide sequence may be a bridging moiety non-covalently bound to an antibody, a fluorescent moiety or another moiety which is detectable by appropriate means. Alternatively, the bridging moiety could be a lectin, to which is bound another moiety that is detectable by appropriate means.
There are a wide variety of signalling moieties and bridging moieties that may be employed in labels for covalent attachment or non-covalent binding to polynucleotide sequences useful as probes in analyte detection systems. They include both a wide variety of radioactive and non-radioactive signalling moieties and a wide variety of non-radioactive bridging moieties. All that is required is that the signalling moiety provide a signal that may be detected by appropriate means and that the bridging moiety, if any, be characterized by the ability to attach covalently or to bind non-covalently to the sequence and also the ability to combine with a signalling moiety.
Radioactive signalling moieties and combinations of various bridging moieties and radioactive signalling moieties are characterized by one or more radioisotopes such as 32P, 131I, 14C, 3H, 60Co, 59Ni, 63Ni and the like. Preferably, the isotope employed emits β or γ radiation and has a long half life. Detection of the radioactive signal is then, most usually, accomplished by means of a radioactivity detector, such as exposure to a film.
The disadvantages of employing a radioactive signalling moiety on a probe for use in the identification of analytes are well known to those skilled in the art and include the precautions and hazards involved in handling radioactive material, the short life span of such material and the correlatively large expenses involved in use of radioactive materials.
Non-radioactive signalling moieties and combinations of bridging moieties and non-radioactive signalling moieties are being increasingly used both in research and clinical settings. Because these signalling and bridging moieties do not involve radioactivity, the techniques and labelled probes using them are safer, cleaner, generally more stable when stored, and consequently cheaper to use. Detection sensitivities of the non-radioactive signalling moieties also are as high or higher than radio-labelling techniques.
Among the presently preferred non-radioactive signalling moieties or combinations of bridging/signalling moieties useful as non-radioactive labels are those based on the biotin/avidin binding system. [P. R. Langer et al., “Enzymatic Synthesis Of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes”, Proc. Natl. Acad. Sci. USA, 78, pp. 6633–37 (1981); J. Stavrianopoulos et al., “Glycosylated DNA Probes For Hybridization/Dection Of Homologous Sequences”, presented at the Third Annual Congress For Recombinant DNA Research (1983); R. H. Singer and D. C. Ward, “Actin Gene Expression Visualized In Chicken Muscle Tissue Culture By Using In Situ Hybridization With A Biotinated Nucleotide Analog”, Proc. Natl. Acad. Sci. USA, 79, pp. 7331–35 (1982)]. For a review of non-radioactive signalling and bridging/signalling systems, both biotin/avidin and otherwise, see D. C. Ward et al., “Modified Nucleotides And Methods Of Preparing And Using Same”, European Patent application No. 63879.
The above-referenced U.S. Patent Application Ser. No. 06/255,223 was abandoned in favor of continuation application, U.S. Patent Application Ser. No. 06/496,915, filed on May 23, 1983, now U.S. Pat. No. 4,711,955. A related divisional application of the aforementioned Ser. No. 06/496,915 was filed (on Dec. 8, 1987) as U.S. Patent Application Ser. No. 07/130,070 and issued on Jul. 12, 1994 as U.S. Pat. No. 5,328,824. Two related continuation applications of the aforementioned Ser. No. 07/130,070 were filed on Feb. 26, 1992 (as Ser. No. 07/841,910) and on May 20, 1992 as (Ser. No. 07/886,660). The aforementioned applications, Ser. No. 07/886,660 and Ser. No. 07/841,910, issued as U.S. Pat. Nos. 5,449,767 and 5,476,928 on Sep. 12, 1995 and Dec. 19, 1995, respectively. The above-referenced U.S. Patent Application Ser. No. 06/391,440, filed on Jun. 23, 1982, was abandoned in favor of U.S. Patent Application Ser. No. 07/140,980, filed on Jan. 5, 1988, the latter now abandoned. Two divisional applications of the aforementioned Ser. No. 07/140,980, U.S. Patent Applications Ser. Nos. 07/532,704 (filed on Jun. 4, 1990 for “Base Moiety Labeled Detectable Nucleotide”) and 07/567,039 (filed on Aug. 13, 1990 for “Saccharide Specific Binding System Labeled Nucleotides”) issued as U.S. Pat. Nos. 5,241,060 (Aug. 31, 1993) and 5,260,433 (Nov. 9, 1993), respectively. The disclosures of the above-identified PNAS article (P. R. Langer et al., “Enzymatic Synthesis of Biotin-Labeled Polynucleotides: Novel Nucleic Acid Affinity Probes,” Proc. Natl. Acad. Sci.(USA) 78:6633–6637 (1981) and U.S. Pat. Nos. 4,711,955, 5,328,824, 5,449,767, 5,476,928, 5,241,060, 5,260,433, and 4,358,535 are herein incorporated and made part of this disclosure.
Generally, the signalling moieties employed in both radioactive and non-radioactive detection techniques involve the use of complex methods for determining the signalling event, and/or supply only an unquantitable positive or negative response. For example, radioactive isotopes must be read by a radioactivity counter; while signalling moieties forming insoluble “signals”, i.e., precipitates, certain fluorescers, and the like [see, e.g., David et al., U.S. Pat. No. 4,376,100] only provide detection not quantitation of the analyte present in the tested sample.
One step toward facilitating rapid and efficient quantitation as well as detection of the hybridization event was the work of Heller et al. in European Patent Applications No. 70685 and 70687 which describe the use of a signalling moiety which produces a soluble signal for measurable detection by a spectrophotometer. These European patent applications disclose the use of two different probes complementary to different portions of a gene sequence, with each probe being labelled at the end which will abut the other probe upon hybridization. The first probe is labelled with a chemiluminescent complex that emits lights of a specific wavelength. The second probe is labelled with a molecule that emits light of a different wavelength measurable by spectrophotometry when excited by the proximity of the first signalling moiety. However, this technique is performed in solution and can generate false positive results in the absence of the analyte if the two probes happen to approach too closely in solution and react with each other.
Similarly, U.K. Patent Application 2,019,408A, published Oct. 31, 1979, discloses a method for detecting nucleic acid sequences in solution by employing an enzyme-labelled RNA or DNA probe which, upon contact with a chromogen substrate, provides an optically readable signal. The analytes may be separated from contaminants prior to hybridization with the probe, or, alternatively, the hybrid probe-analyte may be removed from solution by conventional means, i.e., centrifugation, molecular weight exclusion, and the like. Like Heller's technique, this method is performed in solution.
There remains therefore a need in the art for a reliable, simple and quantifiable technique for the detection of analytes of interest in biological and non-biological samples.